We will develop the basic tools for phylogenetic studies of Mycobacterium tuberculosis (Mtb), and then investigate hypotheses fundamental to understanding the evolution and strain differentiation of the Mtb species. These issues are important for understanding the biology and epidemiology of many bacterial pathogens. A microbial "species" often encompasses related strains or clones with distinct genotypic and phenotypic characteristics. Studies of epidemiology, pathogenesis, and immunity may depend on the ability to identify and classify strains into larger related groups. Mtb has been recalcitrant to this type of analysis because of its relatively low level of genetic polymorphism. We identified a large number of single nucleotide polymorphisms (SNPs) in Mtb based on a comparison of two complete Mtb genome sequences. Preliminary investigations demonstrated that these SNPs are highly polymorphic in clinical Mtb strains and are excellent phylogenetic markers. We then sequenced (to 5X coverage) the genome of a third clinical Mtb strain. Preliminary analysis confirms that comparisons with this strain provide an additional set of novel SNP markers. We also developed a new method for high-throughput SNP analysis that is simple and inexpensive. We now propose to develop a SNP-based phylogenetic model of the Mtb species, and to investigate basic hypotheses about Mtb phylogenetics, evolution, and epidemiology. Our aims are 1) To determine the minimum number and type of SNP markers required to define a high-resolution phylogenetic tree of the Mtb species, and to construct such a tree with a minimal SNP set. This aim will discover novel and informative SNPs by performing a six-way comparison between the genomes of H3 7Rv, CDCI551, the 210 strain, and M bovis. 2) To investigate basic phylogenetic hypotheses including whether the SNP-based phylogenetic data is tree-like, clock-like, and reproducible. We will also investigate if the Mtb species contains a common origin and undergoes introgression or lateral gene exchange; and we will investigate the evolutionary relationships between different Mtb strains and M. bovis. 3) To use the SNP-based phylogeny to investigate other analytic systems including IS6l 10 and VNTR-based DNA fingerprinting; and to provide insights into TB molecular epidemiology and evolution. 4) To identify and validate an informative set of SNPs for worldwide evolutionary and epidemiological analyses.